Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties.

OBJECTIVE: Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. APPROACH AND RESULTS: Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. CONCLUSIONS: The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.

Human mast cells arise from a common circulating progenitor.

BACKGROUND: Human tissue mast cells (MCs) have the potential to express several neutral granule proteases, which are the most precise markers of the phenotypic heterogeneity of MCs. However, the full extent of such heterogeneity is limited by the fact that MCs containing either tryptase only or tryptase and chymase have long been considered to be the sole MC phenotypes. Moreover, the potential developmental relationship between human MCs of different protease phenotypes has remained a matter of dispute. OBJECTIVE: We attempted to define how human MCs with different protease phenotypes relate to their circulating progenitors. METHODS: MCs were generated from human peripheral blood-derived CD34(+) progenitors in the presence of kit ligand (KITLG) and the cytokines IL-3, IL-9, and IL-6 under serum-free conditions, or by KITLG alone in the presence or absence of serum. The expression of chymase, carboxypeptidase A3, cathepsin G, granzyme B, and the tryptases derived from the TPSAB1, TPSB2, TPSD1, and TPSG1/PRSS31 genes were determined weekly at the mRNA and/or protein levels. RESULTS: Incubation of CD34(+) progenitors in the presence of KITLG and the cytokines IL-3, IL-9, and IL-6 promoted the development of a single population of MCs with a uniform tryptase(+), chymase(+), CPA3(+), cathepsin G(+), and granzyme B(+) phenotype. Interestingly, the presence of KITLG alone was sufficient to induce the expression of all the above proteases. CONCLUSION: All circulating human MC progenitors have the potential to differentiate into MCs expressing the complete panel of neutral granule proteases, implying that human MCs originate from a common MC-committed progenitor.

Effects of probiotic Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS supplementation on intestinal and systemic markers of inflammation in ApoE*3Leiden mice consuming a high-fat diet.

A high-fat diet disturbs the composition and function of the gut microbiota and generates local gut-associated and also systemic responses. Intestinal mast cells, for their part, secrete mediators which play a role in the orchestration of physiological and immunological functions of the intestine. Probiotic bacteria, again, help to maintain the homeostasis of the gut microbiota by protecting the gut epithelium and regulating the local immune system. In the present study, we explored the effects of two probiotic bacteria, Lactobacillus rhamnosus GG (GG) and Propionibacterium freudenreichii spp. shermanii JS (PJS), on high fat-fed ApoE*3Leiden mice by estimating the mast cell numbers and the immunoreactivity of TNF-α and IL-10 in the intestine, as well as plasma levels of several markers of inflammation and parameters of lipid metabolism. We found that mice that received GG and PJS exhibited significantly lower numbers of intestinal mast cells compared with control mice. PJS lowered intestinal immunoreactivity of TNF-α, while GG increased intestinal IL-10. PJS was also observed to lower the plasma levels of markers of inflammation including vascular cell adhesion molecule 1, and also the amount of gonadal adipose tissue. GG lowered alanine aminotransferase, a marker of hepatocellular activation. Collectively, these data demonstrate that probiotic GG and PJS tend to down-regulate both intestinal and systemic pro-inflammatory changes induced by a high-fat diet in this humanised mouse model.

Serum amyloid A activates the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway.

Serum amyloid A (SAA) is an acute-phase protein, the serum levels of which can increase up to 1000-fold during inflammation. SAA has a pathogenic role in amyloid A-type amyloidosis, and increased serum levels of SAA correlate with the risk for cardiovascular diseases. IL-1ß is a key proinflammatory cytokine, and its secretion is strictly controlled by the inflammasomes. We studied the role of SAA in the regulation of IL-1ß production and activation of the inflammasome cascade in human and mouse macrophages, as well as in THP-1 cells. SAA could provide a signal for the induction of pro-IL-1ß expression and for inflammasome activation, resulting in secretion of mature IL-1ß. Blocking TLR2 and TLR4 attenuated SAA-induced expression of IL1B, whereas inhibition of caspase-1 and the ATP receptor P2X(7) abrogated the release of mature IL-1ß. NLRP3 inflammasome consists of the NLRP3 receptor and the adaptor protein apoptosis-associated speck-like protein containing CARD (a caspase-recruitment domain) (ASC). SAA-mediated IL-1ß secretion was markedly reduced in ASC(-/-) macrophages, and silencing NLRP3 decreased IL-1ß secretion, confirming NLRP3 as the SAA-responsive inflammasome. Inflammasome activation was dependent on cathepsin B activity, but it was not associated with lysosomal destabilization. SAA also induced secretion of cathepsin B and ASC. In conclusion, SAA can induce the expression of pro-IL-1ß and activation of the NLRP3 inflammasome via P2X(7) receptor and a cathepsin B-sensitive pathway. Thus, during systemic inflammation, SAA may promote the production of IL-1ß in tissues. Furthermore, the SAA-induced secretion of active cathepsin B may lead to extracellular processing of SAA and, thus, potentially to the development of amyloid A amyloidosis.

Lipid-Laden Macrophages and Inflammation in Atherosclerosis and Cancer: An Integrative View.

Atherosclerotic arterial plaques and malignant solid tumors contain macrophages, which participate in anaerobic metabolism, acidosis, and inflammatory processes inherent in the development of either disease. The tissue-resident macrophage populations originate from precursor cells derived from the yolk sac and from circulating bone marrow-derived monocytes. In the tissues, they differentiate into varying functional phenotypes in response to local microenvironmental stimulation. Broadly categorized, the macrophages are activated to polarize into proinflammatory M1 and anti-inflammatory M2 phenotypes; yet, noticeable plasticity allows them to dynamically shift between several distinct functional subtypes. In atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates within macrophages as cytoplasmic lipid droplets thereby generating macrophage foam cells, which are involved in all steps of atherosclerosis. The conversion of macrophages into foam cells may suppress the expression of given proinflammatory genes and thereby initiate their transcriptional reprogramming toward an anti-inflammatory phenotype. In this particular sense, foam cell formation can be considered anti-atherogenic. The tumor-associated macrophages (TAMs) may become polarized into anti-tumoral M1 and pro-tumoral M2 phenotypes. Mechanistically, the TAMs can regulate the survival and proliferation of the surrounding cancer cells and participate in various aspects of tumor formation, progression, and metastasis. The TAMs may accumulate lipids, but their type and their specific roles in tumorigenesis are still poorly understood. Here, we discuss how the phenotypic and functional plasticity of macrophages allows their multifunctional response to the distinct microenvironments in developing atherosclerotic lesions and in developing malignant tumors. We also discuss how the inflammatory reactions of the macrophages may influence the development of atherosclerotic plaques and malignant tumors, and highlight the potential therapeutic effects of targeting lipid-laden macrophages in either disease.

Lipid body formation during maturation of human mast cells.

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.

Acidic extracellular environments strongly impair ABCA1-mediated cholesterol efflux from human macrophage foam cells.

OBJECTIVE: In the deep microenvironments of advanced human atherosclerotic lesions, the intimal fluid becomes acidic. We examined the effect of an acidic extracellular pH on cholesterol removal (efflux) from primary human macrophages. METHODS AND RESULTS: When cholesterol efflux from acetyl-low-density lipoprotein-loaded macrophages to various cholesterol acceptors was evaluated at pH 7.5, 6.5, or 5.5, the lower the pH the more was cholesterol efflux reduced. The reduction of efflux to lipid-free apolipoprotein A-I was stronger than to high-density lipoprotein(2) or to plasma. Cholesterol efflux to every acceptor was severely compromised also at neutral pH when the macrophages had been loaded with cholesterol at acidic pH, or when both loading and efflux were carried out at acidic pH. Compatible with these observations, the typical upregulation of ABCA1 and ABCG1 mRNA levels in macrophages loaded with cholesterol at neutral pH was rapidly attenuated in acidic medium. The secondary structure of apolipoprotein A-I did not changed over the pH range studied, supporting the notion that the inhibitory effect of acidic pH on cholesterol efflux rather impaired the ability of the foam cells to facilitate ABCA1-mediated cholesterol release. Secretion of apolipoprotein E from the foam cells was fully inhibited when the pH was 5.5, which further reduced cholesterol efflux. CONCLUSIONS: An acidic pH reduces cholesterol efflux via different pathways and particularly impairs the function of the ABCA1 transporter. The pH-sensitive function of human macrophage foam cells in releasing cholesterol may accelerate lipid accumulation in deep areas of advanced atherosclerotic plaques where the intimal fluid is acidic.

Vascular endothelial growth factor-secreting mast cells and myofibroblasts: a novel self-perpetuating angiogenic pathway in aortic valve stenosis.

OBJECTIVE: To examine the proangiogenic potential of myofibroblasts and mast cells, 2 types of cells present in human aortic valves. METHODS AND RESULTS: Aortic valve stenosis is an active atheroinflammatory disease, characterized by the accumulation of inflammatory cells and the neovascularization of the valves. A total of 85 stenotic valves and 20 control valves were obtained during valve replacement surgery. The results of immunohistochemistry analysis revealed stenotic aortic valves that contained 3 types of neovessels: small microvessels, medium microvessels, and organized arterioles. The distribution density of the neovessels was significantly higher in stenotic valves than in control valves (P<0.001) and correlated positively with valvular calcification gradus (r=0.26, P=0.02) and mast cell density (r=0.38, P<0.001). In the neovascularized areas of stenotic aortic valves, mast cells contained vascular endothelial growth factor and were degranulated, indicating their activation. The stimulation of cultured myofibroblasts derived from aortic valves with a mast cell-preconditioned medium, hypoxic culture conditions, or tobacco smoke all induced vascular endothelial growth factor secretion in the myofibroblasts. Finally, mast cell tryptase was able to degrade the antiangiogenic molecule endostatin in vitro. CONCLUSIONS: Mast cells and myofibroblasts may accelerate the progression of aortic valve stenosis by altering the balance between angiogenic and antiangiogenic factors in the valves, thus promoting valvular neovascularization.

Cholesterol loading suppresses the atheroinflammatory gene polarization of human macrophages induced by colony stimulating factors.

In atherosclerotic lesions, blood-derived monocytes differentiate into distinct macrophage subpopulations, and further into cholesterol-filled foam cells under a complex milieu of cytokines, which also contains macrophage-colony stimulating factor (M-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF). Here we generated human macrophages in the presence of either M-CSF or GM-CSF to obtain M-MØ and GM-MØ, respectively. The macrophages were converted into cholesterol-loaded foam cells by incubating them with acetyl-LDL, and their atheroinflammatory gene expression profiles were then assessed. Compared with GM-MØ, the M-MØ expressed higher levels of CD36, SRA1, and ACAT1, and also exhibited a greater ability to take up acetyl-LDL, esterify cholesterol, and become converted to foam cells. M-MØ foam cells expressed higher levels of ABCA1 and ABCG1, and, correspondingly, exhibited higher rates of cholesterol efflux to apoA-I and HDL2. Cholesterol loading of M-MØ strongly suppressed the high baseline expression of CCL2, whereas in GM-MØ the low baseline expression CCL2 remained unchanged during cholesterol loading. The expression of TNFA, IL1B, and CXCL8 were reduced in LPS-activated macrophage foam cells of either subtype. In summary, cholesterol loading converged the CSF-dependent expression of key genes related to intracellular cholesterol balance and inflammation. These findings suggest that transformation of CSF-polarized macrophages into foam cells may reduce their atheroinflammatory potential in atherogenesis.

Genetic variations of leptin and leptin receptor are associated with body composition changes in response to physical training.

Leptin regulates body weight, metabolism, and tissue adaptations to environmental stressors. We examined the association of single nucleotide polymorphism (SNP) of leptin promoter G-2548A (rs7799039) and leptin receptor Gln223Arg (rs1137101) with body composition, plasma leptin levels, and peak oxygen uptake (VO(2)peak) in response to 8 weeks of physical training in 48 male military conscripts. AA homozygotes of leptin promoter SNP-2548 showed higher body fat and BMI values than G allele carriers. Acute exercise decreased leptin levels in G allele carriers, but increased in AA homozygotes. Physical training significantly decreased BMI values and also a tendency for decreased plasma leptin levels was observed in all subjects. In G allele carriers, BMI loss was mainly due to decreased fat mass, whereas in AA homozygotes due to loss of fat-free mass. Training increased VO(2)peak in all subjects with most prominent effects in G allele carriers. Regarding leptin receptor SNP, there were no statistically significant differences in BMI values between the genotype groups at baseline or after physical training. Our results suggest that physical training-induced alterations in body composition and plasma leptin may be influenced by a genetic variation of leptin promoter but not of leptin receptor.

Alpha-lipoic acid does not alter stress protein response to acute exercise in diabetic brain.

Heat shock proteins (HSPs) are molecular chaperones which may act protective in cerebrovascular insults and peripheral diabetic neuropathy. We hypothesized that alpha-lipoic acid (LA), a natural thiol antioxidant, may enhance brain HSP response in diabetes. Rats with or without streptozotocin-induced diabetes were treated with LA or saline for 8 weeks. Half of the rats were subjected to exhaustive exercise to investigate HSP induction, and the brain tissue was analyzed. Diabetes increased constitutive HSC70 mRNA, and decreased HSP90 and glucose-regulated protein 75 (GRP75) mRNA without affecting protein levels. Exercise increased HSP90 protein and mRNA, and also GRP75 and heme oxygenase-1 (HO-1) mRNA only in non-diabetic animals. LA had no significant effect on brain HSPs, although LA increased HSC70 and HO-1 mRNA in diabetic animals and decreased HSC70 mRNA in non-diabetic animals. Eukaryotic translation elongation factor-2, essential for protein synthesis, was decreased by diabetes and suggesting a mechanism for the impaired HSP response related to translocation of the nascent chain during protein synthesis. LA supplementation does not offset the adverse effects of diabetes on brain HSP mRNA expression. Diabetes may impair HSP translation through elongation factors related to nascent chain translocation and subsequent responses to acute stress.

Acidic extracellular pH promotes accumulation of free cholesterol in human monocyte-derived macrophages via inhibition of ACAT1 activity.

BACKGROUND AND AIMS: In focal areas of advanced human atherosclerotic lesions, the intimal fluid is acidic. An acidic medium impairs the ABCA1-mediated cholesterol efflux from macrophages, so tending to increase their content of free cholesterol, which is then available for esterification by the macrophage enzyme ACAT1. Here we investigated whether low extracellular pH would affect the activity of ACAT1. METHODS: - Human monocyte-derived macrophages were first incubated with acetyl-LDL at neutral and acidic conditions (pH 7.5, 6.5, and 5.5) to generate foam cells, and then the foam cells were incubated with [3H]oleate-BSA complexes, and the formation of [3H]oleate-labeled cholesteryl esters was measured. ACAT1 activity was also measured in cell-free macrophage extracts. RESULTS: - In acidic media, ACAT1-dependent cholesteryl [3H]oleate generation became compromised in the developing foam cells and their content of free cholesterol increased. In line with this finding, ACAT1 activity in the soluble cell-free fraction derived from macrophage foam cells peaked at pH 7, and gradually decreased under acidic pH with a rapid drop below pH 6.5. Incubation of macrophages under progressively more acidic conditions (until pH 5.5) lowered the cytosolic pH of macrophages (down to pH 6.0). Such intracellular acidification did not affect macrophage gene expression of ACAT1 or the neutral CEH. CONCLUSIONS: Exposure of human macrophage foam cells to acidic conditions lowers their intracellular pH with simultaneous decrease in ACAT1 activity. This reduces cholesterol esterification and thus leads to accumulation of potentially toxic levels of free cholesterol, a contributing factor to macrophage foam cell death.

Pro-atherogenic lung and oral pathogens induce an inflammatory response in human and mouse mast cells.

A broad variety of microbes are present in atherosclerotic plaques and chronic bacterial infection increases the risk of atherosclerosis by mechanisms that have remained vague. One possible mechanism is that bacteria or bacterial products activate plaque mast cells that are known to participate in the pathogenesis of atherosclerosis. Here, we show by real-time PCR analysis and ELISA that Chlamydia pneumoniae (Cpn) and a periodontal pathogen, Aggregatibacter actinomycetemcomitans (Aa), both induce a time and concentration-dependent expression and secretion of interleukin 8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1) by cultured human peripheral blood-derived mast cells, but not anti-inflammatory molecules, such as IL-10 or transforming growth factor beta 1 (TGF-beta 1). The IL-8 and MCP-1 responses were immediate, whereas the onset of TNF-alpha secretion was delayed. The Cpn-mediated pro-inflammatory effect was attenuated when the bacteria were inactivated by UV-treatment. Human monocyte-derived macrophages that were pre-infected with Cpn also induced a significant pro-inflammatory response in human mast cells, both in cocultures and when preconditioned media from Cpn-infected macrophages were used. Intranasal and intravenous administration of live Cpn and Aa, respectively induced an accumulation of activated mast cells in the aortic sinus of apolipoprotein E-deficient mice, however, with varying responses in the systemic levels of lipopolysaccharide (LPS) and TNF-alpha. Pro-atherogenic Cpn and Aa induce a pro-inflammatory response in cultured human connective tissue-type mast cells and activation of mouse aortic mast cells in vivo.

Heat shock proteins in diabetes and wound healing.

The heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a highly conserved family of proteins that respond to a wide variety of stress. Although HSPs are among the most abundant intracellular proteins, they are expressed at low levels under normal physiological conditions, and show marked induction in response to various stressors. HSPs function primarily as molecular chaperones, facilitating the folding of other cellular proteins, preventing protein aggregation, or targeting improperly folded proteins to specific pathways for degradation. By modulating inflammation, wound debris clearance, cell proliferation, migration and collagen synthesis, HSPs are essential for normal wound healing of the skin. In this review, our goal is to discuss the role and clinical implications of HSP with respect to skin wound healing and diabetes. The numerous defects in the function of HSPs associated with diabetes could contribute to the commonly observed complications and delayed wound healing in diabetics. Several physical, pharmacological and genetic approaches may be considered to address HSP-directed therapies both in the laboratory and in the clinics.

Diabetes impairs exercise training-associated thioredoxin response and glutathione status in rat brain.

Regular exercise plays an important preventive and therapeutic role in oxidative stress-associated diseases such as diabetes and its complications. Thiol antioxidants including thioredoxin (TRX) and glutathione (GSH) have a crucial role in controlling cellular redox status. In this study, the effects of 8 wk of exercise training on brain TRX and GSH systems, and antioxidant enzymes were tested in rats with or without streptozotocin-induced diabetes. We found that in untrained animals, the levels of TRX-1 (TRX1) protein and activity, and thioredoxin-interacting protein (TXNip) were similar in diabetic and nondiabetic animals. Exercise training, however, increased TRX1 protein in nondiabetic animals without affecting TXNip levels, whereas diabetes inhibited the effect of training on TRX1 protein and also increased TXNip mRNA. In addition, the proportion of oxidized glutathione (GSSG) to total GSH was increased in animals with diabetes, indicating altered redox status and possibly increased oxidative stress. Glutathione peroxidase-1 (GPX1) levels were not affected by diabetes or exercise training, although diabetes increased total GPX activity. Both diabetes and exercise training decreased glutathione reductase (GRD) activity and cytosolic superoxide dismutase (Cu,Zn-SOD) levels. Nevertheless, diabetes or training had no effect on Cu,Zn-SOD mRNA, Mn-SOD protein, total SOD activity, or catalase mRNA, protein, or activity. Our findings suggest that exercise training increases TRX1 levels in brain without a concomitant rise in TXNip, and that experimental diabetes is associated with an incomplete TRX response to training. Increased oxidative stress may be both a cause and a consequence of perturbed antioxidant defenses in the diabetic brain.

Food allergy alters jejunal circular muscle contractility and induces local inflammatory cytokine expression in a mouse model.

BACKGROUND: We hypothesized that food allergy causes a state of non-specific jejunal dysmotility. This was tested in a mouse model. METHODS: Balb/c mice were epicutaneously sensitized with ovalbumin and challenged with 10 intragastric ovalbumin administrations every second day. Smooth muscle contractility of isolated circular jejunal sections was studied in organ bath with increasing concentrations of carbamylcholine chloride (carbachol). Smooth muscle layer thickness and mast cell protease-1 (MMCP-1) positive cell density were assayed histologically. Serum MMCP-1 and immunoglobulins were quantified by ELISA, and mRNA expressions of IFN-gamma, IL-4, IL-6 and TGFbeta-1 from jejunal and ileal tissue segments were analyzed with quantitative real-time PCR. RESULTS: Ovalbumin-specific serum IgE correlated with jejunal MMCP-1+ cell density. In the allergic mice, higher concentrations of carbachol were required to reach submaximal muscular stimulation, particularly in preparations derived from mice with diarrhoea. Decreased sensitivity to carbachol was associated with increased expression of IL-4 and IL-6 mRNA in jejunum. Smooth muscle layer thickness, as well as mRNA of IFN-gamma and TGF-beta1 remained unchanged. CONCLUSION: In this mouse model of food allergy, we demonstrated a decreased response to a muscarinic agonist, and increased levels of proinflammatory IL-6 and Th2-related IL-4, but not Th1-related IFN-gamma mRNAs in jejunum. IgE levels in serum correlated with the number of jejunal MMCP-1+ cells, and predicted diarrhoea. Overall, these changes may reflect a protective mechanism of the gut in food allergy.

A common variation in the promoter region of interleukin-6 gene shows association with exercise performance.

UNLABELLED: Skeletal muscle-derived interleukin-6 (IL-6) is a pleiotropic cytokine which regulates body metabolism during strenuous physical exercise. OBJECTIVE: The effect of a potentially functional single nucleotide polymorphism (SNP) -174G/C of the IL6 gene (rs1800795) promoter was examined on maximal oxygen uptake (VO2max), body mass index (BMI) and plasma IL-6 levels in response to physical training. Fifty four male military conscripts were studied for 8 weeks during their basic training. At weeks 1, 5 and 8, VO2max and anthropometrics were measured, and blood samples collected before and after acute aerobic exercise. Acute exercise increased plasma IL-6 in subjects with genotype CG. Moreover, during the 8-week training period, a tendency for increased plasma IL-6 was observed in subjects with this genotype. VO2max values increased in all genotype groups, but subjects with genotype CG made the greatest gains in VO2max. Training significantly decreased BMI only in subjects with genotype CG. Our findings suggest that the allele C may have an effect on plasma IL-6 response to acute exercise in healthy male subjects. Exercise training has a favourable effect on VO2max and BMI, with the most prominent effects in subjects with genotype CG. Thus we conclude that this SNP may account for individual response to exercise training. Key pointsAllele C of the IL6 promoter SNP -174G/C may have an effect on plasma IL-6 response to acute exercise.All subjects responded to physical exercise, but the improvement in VO2max and decrease in BMI after training are more pronounced in the individuals with genotype CG, hence the IL6 promoter SNP -174G/C may have an influence on training responses.The small number of subjects investigated in the present study warrants further research to confirm these findings in large cohorts.

Genetic variation in ICOS regulates mRNA levels of ICOS and splicing isoforms of CTLA4.

Genetic and functional studies suggest that polymorphism in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) and inducible costimulator (ICOS) genes, both reported to harbour autoimmune susceptibility loci, could regulate the immune activation through affecting their expression and splicing of CTLA4. To address this, we studied expression of CTLA4 and ICOS and the role of polymorphisms in the gene region by measuring the relative amounts of transcripts, including the soluble CTLA4 (sCTLA4) splicing isoform in healthy volunteers. We combined a physiologically relevant in vitro activation for human CD4(+) T lymphocytes and a quantitative RT-PCR. The susceptibility allele CT60G in CTLA4 gene was confirmed to be associated with a decreased amount of sCTLA4, but only in resting cells. During the T cell activation two genetic variants in ICOS gene, IVS1+173T/C and c.1624C/T, affected expression of CTLA4 isoforms and ICOS, respectively. We could not confirm that the level of sCTLA4 is down-regulated following T lymphocyte activation, instead the levels of CTLA4 splicing isoforms correlated to each others. Our results indicate that genetic variation in this gene region regulates the expression of both CTLA4 and ICOS and not only the splicing of sCTLA4 as suggested earlier.

Alpha-lipoic Acid modulates heat shock factor-1 expression in streptozotocin-induced diabetic rat kidney.

Increased oxidative stress and impaired heat shock protein (HSP) synthesis may contribute to diabetic nephropathy. The question of whether 8-week thiol antioxidant alpha-lipoic acid (LA) supplementation modulates HSP response and oxidative stress was studied in the kidney of streptozotocin-induced diabetic (SID) and nondiabetic rats. SID caused a histological mesangial expansion, tubular dilatation, and increased levels of transforming growth factor-beta (TGF-beta), a mediator of glomerulosclerosis. SID increased 4-hydroxynonenal (4-HNE) protein adduct formation, a marker of lipid peroxidation, and heme oxygenase-1 (HO-1), also a marker of oxidative stress. Moreover, SID increased the DNA-binding activity of heat shock factor-1 (HSF-1) and expression of heat shock protein 60 (HSP60). In contrast, LA supplementation partially reversed histological findings of glomerulosclerosis and decreased TGF-beta. LA also increased HSF-1 and decreased HO-1 protein expression, without affecting 4-HNE protein adduct levels. At the mRNA level, LA increased expression of HSF-1, HSP90, and glucose-regulated protein (GRP75) in both control and diabetic animals and HSP72 in SID rats. However, LA supplementation did not affect these HSPs at the protein level. These findings suggest that in addition to its antiglomerulosclerotic effects, LA can induce cytoprotective response in SID.

Increased expression of elastolytic cathepsins S, K, and V and their inhibitor cystatin C in stenotic aortic valves.

OBJECTIVE: To investigate the possible role of elastolytic cathepsins S, K, and V and their endogenous inhibitor cystatin C in adverse extracellular matrix remodeling of stenotic aortic valves. METHODS AND RESULTS: Stenotic aortic valves were collected at valve replacement surgery and control valves at cardiac transplantations. The expression of cathepsins S, K, and V and cystatin C was studied by conventional and real-time polymerase chain reaction and by immunohistochemistry. Total cathepsin activity in the aortic valves was quantified by a fluorometric microassay. When compared with control valves, stenotic valves showed increased mRNA expression of cathepsins S, K, and V (P<0.05 for each) and a higher total cathepsin activity (P<0.001). In stenotic valves, cystatin C mRNA was increased (P<0.05), and cystatin C protein was found particularly in areas with infiltrates of inflammatory cells. Both cathepsin S and cystatin C were present in bony areas of the valves, whereas cathepsin V localized to endothelial cells in areas rich of neovascularization. Incubation of thin sections of aortic valves with cathepsins S, K, and V resulted in severe disruption of elastin fibers, and this cathepsin effect could be blocked by adding cystatin C to the incubation system. CONCLUSIONS: Stenotic aortic valves show increased expression and activity of elastolytic cathepsins S, K, and V. These cathepsins may accelerate the destruction of aortic valvular extracellular matrix, so promoting the progression of aortic stenosis.